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THE PERACETIC ACID-SCHIFF STAIN RAYMOND BANGLE, JR., M.D. Laboratory of Pathology and Pharmacology, National Institute of Arthritis and Metabolic Diseases, National Institutes of Health, U. S. Public Health Service, Bethesda 14, Maryland The organic peracids, such as perbenzoic, performic and peracetic acids, are strong oxidizing agents. It may be considered that they are derived by the interaction of hydrogen peroxide and an organic acid with the formation of an acid peroxide. Thus, the production of peracetic acid may be illustrated as follows: CH.COOH + H2O2 glacial acetic acid * CH3COOOH + peracetic acid H20 The present paper deals first with the known chemical reactions of organic peracids with organic compounds. This provides one with a background for understanding the chemical basis of the peracetic acid-Schiff reaction in tissue sections. This histochemical method then is described, followed by the results of the method in staining selectively various tissue elements. The use of blocking and extraction procedures and of additional staining reactions are presented in order to substantiate the chemical basis of the peracetic acid-Schiff staining in tissue sections. T H E CHEMICAL REACTIONS OP T H E ORGANIC PERACIDS The organic peracids differ from periodic acid not only in their chemical structure but also in their inability to produce cleavage oxidation of vicinal glycols in a polysaccharide complex or of vicinal hydroxy amino groups in hydroxyamino compounds. The chemical reactions of the organic peracids are grouped according to the following classes of organic compounds: unsaturated lipids, amino acids and protein-bound cystine. Unsativrated lipids. Oxidation of unsaturated fatty acids with a peracid results in a variety of reaction products depending upon the nature of the reagent, the conditions of oxidation and the structure of the unsaturated fat.13 17 Oxidation of unsaturated fatty acids occurs preferentially at the double bond. However, the exact mechanism of peracid oxidation is obscured (1) if one does not know if all peracids are structurally alike, if all peracids attack the double bond by adding to it; (2) by furnishing 1 or 2 atoms of oxygen which in turn add to it; or (3) for both reasons; and (4) if the peracid oxidation may be accompanied or followed by secondary hydrolytic cleavage of the double bond. Assuming that all these conditions exist with peracid oxidation of unsaturated acids, the intermediate and final reaction products would include epoxy or oxido acids, peroxido acids, ketohydroxy acids, dihydroxy or polyhydroxy acids, aldehydo acids (semiReceived for publication October 2, 1953. Dr. Bangle is Pathologist. 179 .180 VOL. 24 BANGLE aldehydes) and aldehydes.13 In the case of performic acid oxidation of the double bond, Lillie8 has illustrated the reaction as follows: 0 —HC=CH— + HCO,H 0 / \ CH— —HC > —HC CH— + oxido group OH + H20 HCOOH OH IICH— • —HC dihydroxy group 0—0 and —H C = C H and by cleavage: \- 2 H C 0 3 H > —HC—CH— + 2 HCOOH peroxido group —HC=0 + aldehyde 0=CH— It should be emphasized that this reaction probably is oversimplified and should not be construed as a general reaction for all unsaturated lipids. However, there is evidence that the reaction does occur in certain instances under specific conditions, and it is the only plausible explanation for the mechanism of the peracetic acid-Schiff staining in tissue sections. By contrast, periodic acid, as usually employed histochemically, is capable of producing cleavage oxidation of dihydroxy or polyhydroxy acids to yield aldehydes, though it is incapable of attacking the ethylenic group per se. Amino acids. Toennies and Homiller20 showed that performic acid reacts only with tryptophan, methionine and cystine, among the amino acids. The mechanism of the reaction in the case of tryptophan is not known. Methionine is oxidized to the sulfone level, whereas cystine is oxidized to 2 molecules of cysteic acid. Protein-bound cystine. Sanger18 has used performic acid to oxidize crystalline insulin in order to split the disulfide bridges of the cystine residues. This procedure made possible, on the basis of solubility, the separation of 2 distinct types of polypeptide chains in which the cystine residues had been converted to cysteic acid residues. Alexander, Fox and Hudson1 investigated the oxidation products of the disulfide bond in degreased wool keratin. They found, on the basis of cation-exchange experiments, that on oxidation of wool with peracetic acid, free sulfonic acid groups were not produced. However, on subsequent acid hydrolysis, cysteic acid was produced almost quantitatively. To explain this, they postulate the formation of an intermediate combined heterocyclic mixed imide of a carboxylic and sulfonic acid, respectively. Further data obtained by the same authors provide evidence that a limited oxidation product from wool cystine (possibly a sulfoxide) was formed by a side reaction. There is no mention that sulfinic acids or aldehydes were produced. FEB. 1954 181 PERACETIC ACID-SCHIFF STAIN THE PERACETIC ACID-SCHIFF METHOD The performic acid and the peracetic acid-Schiff methods were introduced independently by Pearse16 and by Lillie.8 The performic acid and the peracetic acid solutions used by Pearse contained an excess of formic acid and of acetic anhydride, respectively. It has been observed that the excess acid in these solutions occasionally is detrimental to tissue sections, causing swelling and dissolution of certain structures and loss of sections from the slide.2 Lillie8, 9 used solutions of performic acid and of peracetic acid prepared according to Greenspan's specifications.6 These solutions contained an excess of hydrogen peroxide and did not result in loss of sections even at 16 hours. Though performic acid and peracetic acid are equally satisfactory for the histochemical reaction, the use of peracetic acid has one definite advantage. Greenspan's peracetic acid reagent is more stable than his performic acid reagent. The latter should be prepared fresh for each batch of sections, whereas peracetic acid is effective for 1, 2 or more weeks depending upon the amount of use. Following oxidation in peracetic acid at room temperature, the tissue sections are washed for 10 minutes in running tap water and then transferred to standard "cold Schiff" reagent manufactured according to Lillie's specifications.7 Three rinses in 0.5 per cent aqueous Na 2 S 2 05 totaling 5 minutes are used directly after the 10 minutes of Schiff treatment. The sections then are washed in water, dehydrated, cleared and mounted in balsam or polystyrene. In this procedure, nuclei usually are rendered Schiff-positive owing to a Feulgen-type nucleal reaction. This source of confusion is overcome readily by use of an iron hematoxylin counterstain. The time interval for oxidation of tissue sections apparently is not critical. Usually 1 or 2 hours are employed, though 5 to 10 minutes often are equally effective. Prolonging the oxidation interval beyond 6 hours may lead to a gradual decrease in intensity of the Schiff coloration, possibly due to destruction of aldehyde groups. The Schiff reaction step should be limited to 10 or 15 minutes. Within this interval, the Schiff reagent stains, so far as is known, only free aldehyde groups. Ketones in vitro occasionally give a typical positive reaction with Schiff's reagent, though the time required for the color change is longer than that with soluble or insoluble aldehydes.3 APPLICATION OF THE PERACETIC ACID-SCHIFF METHOD A positive peracetic acid-Schiff reaction in tissue elements is indicated by a deep pink, red or purple-red color, providing that these elements are not stained similarly by 10 minutes of Schiff treatment without prior oxidation. The nuclear staining has been mentioned above and is based upon the principle of the Feulgen nucleal reaction. Tissue elements that have been examined in this laboratory and found to be stained more or less consistently by the peracetic acid-Schiff method are: myelin sheaths; red blood cells; retinal rod acromere lipid; hard keratin (hair shafts, nails); lipid granules within the epithelium of eccrine sweat 182 BANGLE VOL. 24 glands; specific lipid granules (lipofuscin) within the ovary, testis and adrenal gland; subcutaneous fat cells; and ceroid of the choline-deficiency hepatic cirrhosis of rats. All the tissue structures listed are stained both by the peracetic acid-Schiff method and by oil-soluble dyes, except hard keratin, which is not sudanophilic at room temperature. Paraffin sections may be used providing the lipid material resists extraction during dehydration. The above tissue structures, after staining with oil-soluble dyes, can be decolorized readily by appropriate dye solvents and restained in undiminished amounts, providing that the lipid remains insoluble, in the solvents employed. This indicates that the material stained was, at least in part, lipid. Furthermore, on the basis of chemical analyses, certain of the above tissue structures (ceroid, myelin, hair shafts and subcutaneous fat) are known to contain lipids possessing a varying degree of unsaturation. In view of what has been stated before in regard to the chemical reactions of organic peracids, and in view of the selectivity of the Schiff reagent for aldehyde groups, if limited to 10 minutes' use, it seems likely that the peracetic acid-Schiff reaction in the tissue elements listed is due to oxidation of unsaturated lipids with the production of insoluble aldehyde groups and other products. To substantiate this, it is necessary to employ specific blocking procedures, extraction procedures, and additional histochemical methods. THE USE OF SPECIFIC BLOCKING PROCEDURES 1. Halogenation. Chlorine and bromine add readily to unsaturated acids to yield saturated compounds. There are several factors that control the rate and completeness of the reaction.4 For this reason, the failure of halogenation in blocking the peracetic acid-Schiff staining of a tissue structure may not mean that unsaturated lipid is absent. However, the finding that halogenation prevents the subsequent staining, whereas the solvent for the halogen does not, is strong presumptive evidence that unsaturated lipid is present. For the procedure of halogenation, treatment of tissue sections in bromine:carbon tetrachloride (1:39 volume dilution) at room temperature for 1 to 2 hours has been employed in this laboratory. This procedure blocks completely the subsequent peracetic acidSchiff reaction of myelin sheaths, retinal rod acromere lipid, hair shafts, lipid granules within the eccrine sweat gland epithelium and ceroid. The bromine solvent alone does not affect the subsequent peracetic acid-Schiff staining of these structures, indicating that the reaction is not based upon dissolution of the lipid material in carbon tetrachloride. 2. Blocking of carbonyl groups, particularly aldehyde groups. Though it is probable that a 10-minute Schiff reaction in tissue elements is not due to reactive groups other than aldehydes, the fact that carbonyl blocking reagents abolish the subsequent Schiff reaction is confirmatory evidence. The blocking reagents include phenylhydrazine, aniline chloride and dimedone. A 5 per cent aqueous solution of phenylhydrazine-HCl condenses readily with aldehydes and many ketones at room temperature. 6 An M / l aqueous solution of aniline chloride condenses more readily with aldehydes than with ketones at room temperature. 6 FEB. 1954 PERACETIC ACID-SCHIFF STAIN 183 A saturated solution of dimedone in water reacts only with aldehydes.19 The procedure for blocking is as follows: tissue sections are oxidized in peracetic acid, washed in water, treated in 1 of the blocking reagents at room temperature for varying time intervals, washed in water and then transferred to the Schiff reagent. That the Schiff coloration in a tissue element is prevented by the prior use of one or all of the blocking reagents, but not by the aqueous solvent alone, is indicative that carbonyl (aldehyde or ketone) groups are produced as the result of oxidation. The failure of blocking does not mean necessarily that carbonyl groups are absent, but suggests either that such groups are in some manner unavailable to the blocking reagent or that the conditions of blocking are not optimum. The Schiff staining of peracetic acid oxidized myelin sheaths, ceroid and lipid granules within eccrine sweat gland epithelium is abolished readily by application of phenylhydrazine or aniline chloride at room temperature, thus indicating that aldehyde groups are produced as the result of oxidation. The blocking in the case of hard keratin is less effective unless the procedure is carried out under increased temperature. 3. Blocking of hydroxyl and amino groups. Acetylation or benzoylation readily blocks these groups.9 Since vicinal glycols or vicinal hydroxy amino groups are responsible for the periodate Schiff reaction, acetylation or benzoylation blocking procedures would be expected and are known to prevent this histochemical reaction.9 Since these groups are not attacked by peracetic acid oxidation, the blocking procedures would not be expected to affect the peracetic acid-Schiff reaction. In practice, this is found to be so. LIPID EXTRACTION PROCEDURES The solubility of lipids varies markedly according to the nature of the solvent, the temperature of the solvent and the structure of the fat. In addition, fixation of tissue by formalin or chromate fixation, or both, renders certain lipids, such as those in myelin, less soluble. Therefore, the stainability of a particular tissue structure after the use of a lipid-extraction procedure may not mean that the material stained is other than fat. One method for determining if lipids are removed completely from tissue by an extraction procedure is to analyze the extracted residue chemically for fat content. Another method for checking the effectiveness of a lipid-extraction procedure makes use of the application of an oil-soluble dye to tissue before and after extraction. The so-called "fat solvents" include petroleum ether, hexane, diethyl ether, chloroform, benzene, ethanol, methanol, acetone, gasoline, carbon tetrachloride, pyridine and others. These solvents may be combined. Thus, the use of boiling chloroform: methanol (1:1 or 2:1) is an effective extraction procedure and, furthermore, is capable of fixing thin blocks of tissue. The solubility of the lipid in the various tissue structures stained by the peracetic acid-Schiff method varies. Thus, the reactive lipid in the subcutaneous fat cell is extracted readily by ethanol at room temperature, whereas the reactive lipid of ceroid is extremely difficult to extract even by use of boiling solvents. 184 VOL. 24 BANGLE H I S T O C H E M I C A L METHODS F O R FATTY ACID PEROXIDES Peracid oxidation of unsaturated acids results in a variety of products, among which are peroxides. The histochemical demonstration of peroxides, as well as aldehydes, in a tissue element that previously was oxidized by peracetic acid is supportive evidence that such an element contained unsaturated lipid. The aldehyde groups are detected by the Schiff reagent, whereas the peroxides are detected by the methods described below. 1. The ferric ferricyanide reduction test. Lillie and Burtner 10 found that a variety of substances are capable of reducing ferric ferricyanide mixtures to a blue or deep green, often insoluble compound (Prussian blue). Hydrogen peroxide gave a prompt reaction. Cod liver oil and linseed oil reacted within 10 minutes. They suggest that the reaction with polyunsaturated fats may be due to the presence of fatty acid peroxides rather than to the ethylenic groups per se. Peracetic acid oxidation of unsaturated acids accelerates the production of peroxides as compared to noncatalyzed atmospheric oxidative rancidity. For example, fresh ceroid, fresh hair shafts or fresh subcutaneous fat gives a weak (faint blue or spotty) ferric ferricyanide reduction reaction, although after peracetic acid oxidation the same structures are stained intensely (dark blue). A direct reaction occurs in rancid subcutaneous fat. 2. The indophenol blue synthesis test (the Winkler-Schultze or "M-nadi oxidase" reaction)}2 In the presence of atmospheric oxygen, oxidase or peroxide, an insoluble dark blue compound (indophenol blue) is synthesized from a mixture of a-naphthol and dimethyl-p-phenylenediamine-HCl. If the histochemical reaction is carried out under anaerobic conditions or is limited to less than 5 minutes in air, the production of indophenol blue in a lipid material (sudanophilic material) may be considered as being due to the presence of fat peroxides. This is found to occur in the case of fresh ceroid, fresh hair shafts and fresh subcutaneous fat, only after previous oxidation with peracetic acid. A direct reaction occurs in rancid subcutaneous fat. T H E P E R A C E T I C A C I D - S C H I F F R E A C T I O N O F HARD KERATIN This subject is discussed separately because there is controversy in regard to the mechanism of the reaction. As has been mentioned before, peracetic acid oxidation of hard keratin (hair shafts, nails) gives rise to a product which is stained red with Schiff's reagent. Pearse16, 16 believes that this reaction in tissue sections is due to the oxidation of protein-bound cystine with the subsequent formation not only of cysteic acid (alanine-beta-sulfonic) but also of another acid (alanine-beta-sulfinic). The latter is thought by him to be responsible for the positive reaction with Schiff's reagent. Lillie and Bangle11 have shown that Pearse's hypothesis probably is incorrect. This is based upon the experimental results obtained from use of specific blocking methods (e.g., bromination), of prolonged lipid extraction procedures, of reagents capable of oxidizing sulfinic acids, and of in vitro reactions with sulfinic and sulfonic acids and with insulin. Furthermore, there is no chemical evidence that sulfinic acid groups arise from peracetic acid oxidation of wool keratin. 1 It seems unlikely that the peracetic FEB. 1 9 5 4 PERACETIC ACID-SCHIFF STAIN 185 acid-Schiff reaction in hair shafts is due to adsorption of peracetic acid with subsequent oxidative recolorization of the Schiff reagent.2 A possible explanation of the chemical basis of this reaction as applied to hair shafts is that unsaturated substances are attacked, yielding aldehydes, peroxides and probably other products. Nicolaides14 has shown that human hair shafts possess many unsaturated acids, alcohols and sterols, as well as squalene—a highly unsaturated hydrocarbon. The iodine value of the total fat was 50 to 60. The lipid content of hair shafts is extremely difficult to extract completely without destroying the hair. SUMMARY The peracetic acid-Schiff method is based chemically upon the oxidation of double bonds in unsaturated lipids with the production of insoluble aldehyde groups. Epoxides and peroxides also are produced. The aldehydes are detected by the Schiff reagent, whereas the peroxides are detected by the ferric ferricyanide reduction test or by the indophenol blue synthesis test. These reactions are blocked by prior halogenation (bromination) which converts unsaturated acids to saturated compounds. The latter are nonreactive to peracetic acid oxidation. The application of the peracetic acid-Schiff method to various lipid-containing tissue structures is described. REFERENCES 1. ALEXANDER, P : , F O X , M . , AND HUDSON, R . F . : T h e reaction of oxidizing agents with wool. 5. T h e oxidation products of t h e disulphide bond and t h e formation of a sulphonamide in the peptide chain. Biochem. J., 49: 129-138, 1951. 2. BANGLE, R., J R . : Unpublished observations. 3. Ciiu, C. H . U . : A histochemical s t u d y of staining t h e axis cylinder with fuehsinsulfurous acid (Schiff'$ reagent). Anat. R e c , 108: 723-745, 1950. 4. D E U E L , H . J., J R . : T h e Lipids: Their Chemistry and Biochemistry. E d . 1. New York: Interscience Publishers, I n c . , p p . 153-155, 1951. 5. GREENSPAN, F . P . : T h e convenient preparation of per-acids. J . Am. Chem. S o c , 68: 907, 1946. 6. HICKINBOTTOM, W. J . : Reactions of Organic Compounds. E d . 2. London: Longmans, Green & Co., 1948, 481 p p . 7. L I L L I E , R . D . : Simplification of t h e manufacture of Schiff reagent for use in histochemical procedures. Stain Technol., 26: 163-165, 1951. 8. L I L L I E , R. D . : Ethylenic reaction of ceroid with performic acid and Schiff reagent. Stain Technol., 27: 37-45, 1952. 9. L I L L I E , R . D . : Histopathologic Technic. E d . 2. New Y o r k : Blakiston Co., scheduled to appear in J a n . , 1954. 10. L I L L I E , R . D . , AND BURTNER, H . J . : T h e ferric ferricyanide reduction test in histochemistry. J . Histochem. & Cytochem., 1: 87-92, 1953. 11. L I L L I E , R. D . , AND B A N G L E , R., J R . : Manuscript in p r e p a r a t i o n . 12. LISON, L . : Histochiniie et Cytochimie Animales. E d . 2, P a r i s : Gauthier-Villars, p p . 403-106, 1953. 13. MARKLEY, K . S.: F a t t y Acids: Their Chemistry and Physical Properties. E d . 1. New York: Interscience Publishers, I n c . , 1947, p p . 387-477. 14. NICOLAIDES, N . : Personal communication. 15. P E A R S E , A. G. E . : T h e histochemical demonstration of keratin by methods involving selective oxidation. Quart. J. Micr. So., 92: 393-402, 1951. 16. P E A R S E , A. G. E . : Histochemistry, Theoretical and Applied. E d . 1. Boston: Little, Brown and Co., 1953. 17. RALSTON, A. W . : F a t t y Acids and Their Derivatives. New Y o r k : John Wiley & Sons, Inc., p p . 917-920, 1948. 18. SANGER, F . : Fractionation of oxidized insulin. Biochem. J., 44: 126-128, 1949. 19. SMITH, G. F . : Analytical Applications of Periodic Acid (HsIOo) and Iodic Acid (HIO3) and Their Salts. E d . 5, Champaign, 111.: T h e Garrard Press. 1950. 20. T O E N N I E S , G., AND HOMILLER, R. P . : T h e oxidation of amino acids by hvdrogen peroxide in formic acid. J. Am. Chem. S o c , 64: 3054-3056, 1942.